Flagellin Glycosylation in Pseudomonas aeruginosa PAK Requires the O-antigen Biosynthesis Enzyme WbpO

Miller, Wayne L.; Matewish, Mauricia J.; McNally, David J.; Ishiyama, Noboru; Anderson, Erin M.; Brewer, Dyanne; Brisson, Jean‐Robert; Berghuis, Albert M.; Lam, Joseph S.

doi:10.1074/jbc.m708894200

Cited by 41 publications

(44 citation statements)

References 37 publications

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“…Reactions were performed in triplicate, and results were averaged to calculate the percent conversion for each enzyme with each substrate. For comparison to theoretical reaction products, known standards were obtained from Sigma-Aldrich (UDP-D-GlcA) or through enzymatic synthesis using His 6 -WbpA or His 6 -WbpO according to established methods (UDP-D-GlcNAcA and UDP-D-GalNAcA) (30).…”

Section: Methodsmentioning

confidence: 99%

“…The initial enzyme of the pathway, WbpA, is a 6-dehydrogenase that converts UDP-D-GlcNAc to UDP-N-acetylglucosaminuronic acid (UDP-D-GlcNAcA) (31). Another 6-dehydrogenase, WbpO of P. aeruginosa serotype O6, has 53% similarity to WbpA and has been shown to convert UDP-D-GalNAc to UDP-D-GalNAcA for use in the O antigen but is also capable of converting UDP-D-GlcNAc to UDP-DGlcNAcA (30,48). In P. aeruginosa PAK (serotype O6), the WbpO enzyme is required for both O antigen biosynthesis and flagellin glycosylation (30).…”

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confidence: 99%

“…Another 6-dehydrogenase, WbpO of P. aeruginosa serotype O6, has 53% similarity to WbpA and has been shown to convert UDP-D-GalNAc to UDP-D-GalNAcA for use in the O antigen but is also capable of converting UDP-D-GlcNAc to UDP-DGlcNAcA (30,48). In P. aeruginosa PAK (serotype O6), the WbpO enzyme is required for both O antigen biosynthesis and flagellin glycosylation (30). The B-band O antigen biosynthesis cluster of P. aeruginosa serotype O6 contains wbpO followed by wbpP (3), which encodes a 4-epimerase that can catalyze the reversible conversion of UDP-D-GlcNAc to UDP-D-GalNAc or UDP-D-GlcNAcA to UDP-D-GalNAcA (8,30).…”

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confidence: 99%

“…In P. aeruginosa PAK (serotype O6), the WbpO enzyme is required for both O antigen biosynthesis and flagellin glycosylation (30). The B-band O antigen biosynthesis cluster of P. aeruginosa serotype O6 contains wbpO followed by wbpP (3), which encodes a 4-epimerase that can catalyze the reversible conversion of UDP-D-GlcNAc to UDP-D-GalNAc or UDP-D-GlcNAcA to UDP-D-GalNAcA (8,30). Despite both enzymes being bifunctional, data from kinetic analysis of WbpO and equilibrium analysis of WbpP suggested a preference in vivo for WbpO to work first, converting UDP-D-GlcNAc to UDP-DGlcNAcA, followed by WbpP, converting UDP-D-GlcNAcA to UDP-D-GalNAcA (30).…”

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confidence: 99%

“…The B-band O antigen biosynthesis cluster of P. aeruginosa serotype O6 contains wbpO followed by wbpP (3), which encodes a 4-epimerase that can catalyze the reversible conversion of UDP-D-GlcNAc to UDP-D-GalNAc or UDP-D-GlcNAcA to UDP-D-GalNAcA (8,30). Despite both enzymes being bifunctional, data from kinetic analysis of WbpO and equilibrium analysis of WbpP suggested a preference in vivo for WbpO to work first, converting UDP-D-GlcNAc to UDP-DGlcNAcA, followed by WbpP, converting UDP-D-GlcNAcA to UDP-D-GalNAcA (30). Thus, homologs of either WbpA or WbpO are theoretically capable of providing the required 6-dehydrogenation of UDP-D-GlcNAc to initiate the UDP-D-ManNAc3NAcA biosynthesis pathway.…”

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Biosynthesis of a Rare Di-N-Acetylated Sugar in the Lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis Occurs via an Identical Scheme despite Different Gene Clusters

et al. 2008

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Pseudomonas aeruginosa and Bordetella pertussis produce lipopolysaccharide (LPS) that contains 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA). A five-enzyme biosynthetic pathway that requiresWbpA, WbpB, WbpE, WbpD, and WbpI has been proposed for the production of this sugar in P. aeruginosa, based on analysis of genes present in the B-band LPS biosynthesis cluster. In the analogous B. pertussis cluster, homologs of wbpB to wbpI were present, but a putative dehydrogenase gene was missing; therefore, the biosynthetic mechanism for UDP-D-ManNAc3NAcA was unclear. Nonpolar knockout mutants of each P. aeruginosa gene were constructed. Complementation analysis of the mutants demonstrated that B-band LPS production was restored to P. aeruginosa knockout mutants when the relevant B. pertussis genes were supplied in trans. Thus, the genes that encode the putative oxidase, transaminase, N-acetyltransferase, and epimerase enzymes in B. pertussis are functional homologs of those in P. aeruginosa. Two candidate dehydrogenase genes were located by searching the B. pertussis genome; these have 80% identity to P. aeruginosa wbpO (serotype O6) and 32% identity to wbpA (serotype O5). These genes, wbpO 1629 and wbpO 3150 , were shown to complement a wbpA knockout of P. aeruginosa. Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO 1629 and WbpO 3150 , and mass spectrometry analysis confirmed that the two enzymes are dehydrogenases capable of converting UDP-D-GlcNAc, UDP-D-GalNAc, to a lesser extent, and UDP-D-Glc, to a much lesser extent. Together, these results suggest that B. pertussis produces UDP-D-ManNAc3NAcA through the same pathway proposed for P. aeruginosa, despite differences in the genomic context of the genes involved. Nomenclature for the conceptual division of LPS differs between different organisms. In Bordetella pertussis, an obligate human pathogen and the causative agent of whooping cough, the core oligosaccharide is linked to a structure called the band A trisaccharide. The band A trisaccharide from B. pertussis 1414 is composed of N-acetyl-D-glucosamine (D-GlcNAc), 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA), and 2-acetamido-4-methylaminofucose (FucNAc4NMe) (6). The related species Bordetella bronchiseptica, a pathogen of animals, and Bordetella parapertussis, a pathogen of both humans and animals, also have D-ManNAc3NAcA present in the LPS (33). B. bronchiseptica and B. parapertussis LPS also contains a repeating polysaccharide known as the O antigen (12). The O antigen contains 2,3-diacetamido-2,3-dideoxy-L-galactosamine (L-GalNAc3NAcA) (33), which is thought to be synthesized from UDP-D-ManNAc3NAcA by the enzymes of the wbm gene cluster (21). In B. pertussis, the LPS is not capped with the O antigen due to the deletion of the wbm cluster (33).It is intriguing that the rare di-N-acetylated mannuronic acid sugar residue D-ManNAc3NAcA is present in both P. aeruginosa serogroup O2 and in the LPS of B. pertussis. LPS is an important virulen...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Biosynthesis of a Rare Di-N-Acetylated Sugar in the Lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis Occurs via an Identical Scheme despite Different Gene Clusters

et al. 2008

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Pseudomonas

Westman

Matewish

Lam

2010

Pathogenesis of Bacterial Infections in Animals

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Cofactor‐Driven Cascade Reactions Enable the Efficient Preparation of Sugar Nucleotides

et al. 2022

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Glycosylation is catalyzed by glycosyltransferases using sugar nucleotides or occasionally lipid‐linked phosphosugars as donors. However, only very few common sugar nucleotides that occur in humans can be obtained readily, while the majority of sugar nucleotides that exist in bacteria, plants, archaea, or viruses cannot be synthesized in sufficient quantities by either enzymatic or chemical synthesis. The limited availability of such rare sugar nucleotides is one of the major obstacles that has greatly hampered progress in glycoscience. Herein we describe a general cofactor‐driven cascade conversion strategy for the efficient synthesis of sugar nucleotides. The described strategy allows the large‐scale preparation of rare sugar nucleotides from common sugars in high yields and without the need for tedious purification processes.

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Flagellin Glycosylation in Pseudomonas aeruginosa PAK Requires the O-antigen Biosynthesis Enzyme WbpO

Cited by 41 publications

References 37 publications

Biosynthesis of a Rare Di-N-Acetylated Sugar in the Lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis Occurs via an Identical Scheme despite Different Gene Clusters

Biosynthesis of a Rare Di-N-Acetylated Sugar in the Lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis Occurs via an Identical Scheme despite Different Gene Clusters

Pseudomonas

Cofactor‐Driven Cascade Reactions Enable the Efficient Preparation of Sugar Nucleotides

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